Benjamin L. Turner,* Barbara J. Cade-Menun, and Dale T. Westermann
ABSTRACT The organic P composition of semi-arid arable soils is largely un-
known, but such information is fundamental to understanding P dy- namics in irrigated agriculture. We used solution "P nuclear magnetic resonance (NMR) spectroscopy and phosphatase hydrolysis to charac- terize organic P in semi-arid arable soils from the western USA (or- ganic C 2.0-30.7 g C kg' soil, clay 2-48%, pH 5.2-8.2, CaCO 3 <1-480 g kg -' soil). Total P concentrations ranged from 220 to 1210 mg P kg-' soil, of which between 12 and 45% was extracted with NaOH-EDTA. Inorganic orthophosphate was the dominant P compound, but concen- trations determined by solution "P NMR spectroscopy were consis- tently greater than those determined by molybdate colorimetry. Con- centrations of organic P were relatively small, and were dominated by orthophosphate monoesters (11-130 mg P kg-' soil), with smaller concentrations of orthophosphate diesters (0-7 mg P kg- 1 soil). Pyro- phosphate was present in almost all soils at concentrations up to 14 mg P kg -' soil. Bicarbonate-extractable organic P ranged from 1.7 to 22.8 mg P kg -' soil, of which between 37 and 87% was hydrolyzed by phosphatase enzymes, suggesting its bioavailability. Soil organic P concentrations were positively correlated with mean annual precipi- tation, organic C, clay, and oxalate-extractable metals (Al, Fe, Mn), and negatively correlated with mean annual temperature and soil pH. However, CaCO 3 concentrations were not significantly correlated with any soil property. These results indicate that equilibrium levels of organic P in semi-arid arable soils are controlled by a balance between the physical protection offered by the soil matrix and the suitability of the environment for biological productivity.
I
NFORMATION on the chemical composition of soil or- ganic P is fundamental to understanding plant nutri-
tion and soil biogeochemical cycles. However, organic P remains poorly understood, despite constituting a large proportion of the total P in many soils, and providing a source of P for plant uptake (Magid et al., 1996). In particular, little information exists on the chemical composition of organic P in low organic matter calcare- ous soils, such as those common in the drier regions of the western USA. The semi-arid climate of the western USA means that most agricultural soils are irrigated, and such soils are agronomically important because irri- gated agriculture produces nearly 40% of the total U.S. crop value from only 15% of the total cropped land (Bajwa et al., 1992). Therefore, information on the com- position of organic P and its potential contribution to plant nutrition in these soils must be better understood. Furthermore, current interest in the transfer of soil or- ganic P compounds to watercourses and their impact on water quality (Haygarth and Jarvis, 1999) reinforces
B.L. Turner and D.T. Westermann, USDA-ARS, Northwest Irriga- tion and Soils Research Lab., 3793 N. 3600 E., Kimberly, ID 83341: BJ. Cade-Menun, Dep. of Geological and Environmental Sciences, Stanford Univ., Stanford, CA 94305. Received 1 July 2002. *Corre- sponding author (bturner@nwisrl.ars.usda.gov).
Published in Soil Sci. Soc. Am. J. 67:1168-1179 (2003).
the need to understand the nature of organic P in these soils.
Soil organic P determination has traditionally been hampered by difficulties with the extraction, separation and detection of these often recalcitrant compounds, but analysis by solution 31P NMR spectroscopy obviates many of these problems. When coupled with the single- step NaOH—EDTA extraction procedure (Bowman and Moir, 1993) more organic P can be characterized than by extraction with NaOH alone (Cade-Menun and Preston, 1996). Indeed, more than 90% of the total P in high organic matter soils has been characterized by NaOH- EDTA extraction and solution 31P NMR spectroscopy (Dai et al., 1996; Cade-Menun et al., 2000). Solution 31P NMR spectroscopy is less suitable for the analysis of water or bicarbonate extracts, because the relatively high detection limits confound analysis of samples with low P concentrations (Nanny and Minear, 1997). De- spite this, the organic P in these types of extracts can be characterized by its susceptibility to hydrolysis by phosphatase enzymes (Shand and Smith, 1997; Turner et al., 2002a). This sensitive technique not only provides structural information on the extracted organic P, but also indicates its potential biological availability.
Our objectives were to determine the composition and potential bioavailability of organic P in a range of low organic matter, mainly calcareous, arable soils of the semi-arid western USA, and to investigate how these were related to climate and soil properties.
MATERIALS AND METHODS Soil Sampling, Preparation, and Determination
of Basic Properties Eighteen soils were sampled to 30-cm depth from locations
around the western USA during 2000 (Table 1). Most soils were under irrigated arable cropping, although the Wahpeton soil (No. 18) (soil descriptions are given in Table 1) was not irrigated and the native Portneuf (No. 14) was under non- irrigated sagebrush (Artemisia tridentata Nutt.). The sampling sites were semi-arid, with hot dry summers and cool moist winters. Mean annual temperatures ranged from 5.0°C at Fargo, ND, to 13.8°C at Amarillo, TX. Mean annual precipita- tion ranged from 209 mm at Othello, WA, to 547 mm at Pull- man, WA.
The soils were air-dried (30°C), sieved (<2 mm), and stored at ambient laboratory temperature before analysis. Soil tex- tural information and the concentrations of organic C and CaCO3 were determined by standard methods (Allison and Moodie, 1965; Day, 1965; Nelson and Sommers, 1982). Soil pH was determined on a saturated paste using deionized water (1:1 ratio). Oxalate-extractable Al, Fe, and Mn were deter- mined by extraction with ammonium oxalate/oxalic acid (pH 3.0) for 2 h, followed by detection using inductively coupled plasma atomic-emission spectroscopy (ICP–AES) (Schoumans, 2000).
1168
TURNER ET AL: ORGANIC P COMPOSITION OF SEMI-ARID U.S. SOILS
1169
Table 1. Description, location, and climate information for the 18 western U.S. soils. The soils are ranked in order of their organic C concentrations (see Table 3). Conventionally managed soils (con y) received only inorganic fertilizer, whilst manured soils received a single application of dairy manure several years before sampling.
Soil series Location Description Mean annual temperature
Mean annual precipitation
°C mm
1. Taunton Buhl, ID Coarse-loamy, mixed, superactive, mesic Xeric 9.1 243 Haplodurids
2. Olton Amarillo, TX Fine, mixed, superactive, thermic Aridic Paleustolls 13.8 497 3. Declo Aberdeen, ID Coarse-loamy, mixed, superactive, mesic Xeric 7.2 224
Haplocalcids 4. Warden Othello, WA Coarse-silty, mixed, superactive, mesic Xeric 10.1 209
Haplocambids 5. Amarillo Amarillo, TX Fine-loamy, mixed, superactive, thermic Aridic 13.8 497
Paleustalfs 6. Portneuf (cony subsoil)t Kimberly, ID Coarse-silty, mixed, superactive, mesic Durinodic 8.7 275
Xeric Haplocalcids 7. Greenleaf Parma, ID Fine-silty, mixed, superactive, mesic Xeric 10.2 260
Calciargids 8. Williams Sidney, MT Fine-loamy, mixed, superactive, frigid Typic 5.9 354
Argiustolls 9. Portneuf (manured subsoil)t Kimberly, ID Coarse-silty, mixed, superactive, mesic Durinodic 8.7 275
Xeric Haplocalcids 10. Portneuf (cony) Kimberly, ID Coarse-silty, mixed, superactive, mesic Durinodic 8.7 275
Xeric Haplocalcids 11. Roza Filer, ID Fine, smectitic, mesic Xerertic Haplocambids 8.9 259 12. Portneuf (manured) Kimberly, ID Coarse-silty, mixed, superactive, mesic Durinodic 8.7 275
Xeric Haplocalcids 13. Millville Logan, UT Coarse-silty, carbonatic, mesic Typic Haploxerolls 8.6 420 14. Portneuf (native) Murtaugh, ID Coarse-silty, mixed, superactive, mesic Durinodic 8.7 275
Xeric Haplocalcids 15. Brinegar Fairfield, ID Fine-loamy, mixed, superactive, frigid Pachic Ultic 5.3 391
Argixerolls 16. Palouse Pullman, WA Fine-silty, mixed, superactive, mesic Pachic Ultic 8.3 547
Haploxerolls 17. Labenzo Rexburg, ID Fine-loamy over sandy or sandy-skeletal, mixed,
superactive, frigid Fluventic Haploxerolls 6.4 348
18.Wahpeton Fargo, ND Fine, smectitic, frigid Typic Hapluderts 5.0 494
t Sampled from experimental plots from which topsoil was removed to simulate the effects of soil erosion exposing highly calcareous subsoil (Robbins et al., 1999).
Methodology for Characterizing Soil Phosphorus Soil P was characterized using three procedures (Fig. 1):
1. Determination of total soil P fractions based on diges- tion, ignition, and acid-extraction;
2. Characterization of NaOH-EDTA extractable P by mo- lybdate colorimetry and solution 31 P NMR spectroscopy;
3. Characterization of bicarbonate-extractable P by molyb- date colorimetry and phosphatase hydrolysis.
Determination of Total Soil Phosphorus Fractions
Total soil P was determined by NaOH fusion (Smith and Bain, 1982). Total inorganic and organic P were determined by the ignition method (Saunders and Williams, 1955). Samples of ignited (550°C, 2 h) and unignited soils were extracted for 2 h with 1 M H2SO4. Organic P was calculated as the difference between inorganic P in the ignited and unignited samples, while residual P was calculated as the difference between total soil P and inorganic P in the ignited sample.
NaOH-EDTA Extraction and Solution 35P NMR Spectroscopy
Phosphorus was extracted by shaking 5 g of soil with 100 mL of a solution containing 0.25 M NaOH and 0.05 M EDTA for 16 h at 20°C (Cade-Menun and Preston, 1996). This single- step extraction of organic P is comparable with other alkaline and acid extraction procedures for calcareous soils (Bowman and Moir, 1993). We did not use an acid pre-extraction, be- cause this also extracted some organic P. The NaOH-EDTA extracts were centrifuged at 10 000 x g for 30 min, rapidly
frozen at -80°C, and then freeze-dried over several days. Inorganic P was determined colorimetrically in diluted ex- tracts (1:100) by molybdate reaction with P detection at 880 nm (Murphy and Riley, 1962). The extracts must be diluted at least 20-fold to avoid interference by EDTA in the molybdatc reaction. Total P was determined by ICP-AES and organic P was calculated as the difference between total and inorganic P. The inorganic P fraction is mainly orthophosphate, but can include acid-labile organic and condensed P compounds (Dick and Tabatabai, 1977), while the organic P fraction also includes inorganic polyphosphates (Shand et al., 2000). However, we use the terms inorganic and organic P for clarity. All results are means of three replicate analyses with standard errors (not shown) less than ±5% of the mean value.
Freeze-dried NaOH-EDTA extracts (approximately 1 g) were redissolved in 0.4 mL of 10 M NaOH and 2.6 mL of D 20 and allowed to stand for 30 min with occasional vortexing. Samples were then centrifuged for 20 min at approximately 1500 x g, transferred to 10-mm diam. NMR tubes and stored at 4°C before analysis within 24 h. Solution 31P NMR spectra were obtained using a GE Omega 500 MHz spectrometer equipped with a 10-mm broadband probe (General Electric, Fremont, CA). We used a 90° pulse, 0.68-s acquisition time, and 4.32-s pulse delay. Temperature was regulated at 25°C but it is now recommended that temperature be regulated at 20°C for solution 31 P NMR spectroscopy of soil extracts (Turner et al., 2003). An equal number of scans (8000) was collected for each sample and compounds were identified by their chemical shifts (ppm) relative to an external H 3PO4 stan- dard. General functional classes of P compounds were as fol- lows: inorganic orthophosphate at approximately 6.1 ppm,
1170 SOIL SCI. SOC. AM. J., VOL. 67, JULY-AUGUST 2003
Soil
1 Bicarbonate extraction
NaOH-EDTA extraction
1)Inorganic orthophosphate 2)Orthophosphate monoesters 3)Orthophosphate diesters 4)Pyrophosphate
1)Labile monoesters 2)Phospholipids (diester) 3)Nucleic acids (diester) 4) Inositol hexakisphosphate
(monoester)
1 1
Fig. 1. The fractionation scheme used to characterize soil inorganic and organic P.
orthophosphate monoesters between 3 and 6 ppm, orthophos- phate diesters between –0.5 and 2.0 ppm, pyrophosphate at approximately –4.5 ppm (Turner et al., 2003). The limit of detection using solution 31 P NMR is difficult to quantify, but depends on the P concentration of the freeze-dried extracts and the number of scans acquired. For our study we estimate the limit of detection to be approximately 1 mg P kg - ' soil.
Bicarbonate Extraction and Analysis by Phosphatase Hydrolysis
Bicarbonate-extractable P was determined by shaking 2.5 g of soil with 50 mL of 0.5 M NaHCO 3 (adjusted to pH 8.5 with dilute NaOH) for 30 min (Olsen et al., 1954). Each sample was filtered sequentially through a Whatman No. 42 filter paper (Whatman Ltd., Maidstone, UK) and a 0.2-p.m cellulose acetate syringe filter (Nalgene, Rochester, NY). One-milliliter aliquots were pre-acidified with 0.1 mL of 3 M H 2SO4 (to remove carbonates) and diluted to 5 mL with deionized water. Inorganic P was then determined by adding 1 mL of molybdate reagent and measuring the absorbance after 12 mM at 880 nm. Molybdate-reactive P in bicarbonate extracts includes only inorganic orthophosphate (Coventry et al., 2001). Total P in the extracts was determined by the same procedure fol-
lowing acid-persulphate digestion. Briefly, samples (1 mL) were acidified by adding 0.15 mL of 3 M H 2SO4 and digested with 3.85 mL of 26 mM K2S208 (20 mM final concentration) at 120°C and 100 kPa for 45 mM. Organic P was calculated as the difference between total and inorganic P. Each soil was extracted three times.
For 11 of the 18 soils, bicarbonate extracts were analyzed by phosphatase hydrolysis using the methodology of Turner et al. (2002a), with modifications for bicarbonate extracts, including stronger buffer concentration and the inclusion of a phospholipase. Samples (1 mL) were pre-acidified to remove carbonate by adding 0.1 mL of 3 M H2SO4, then neutralized by adding 0.12 mL of 1 M NaOH. After adding 1 mL of 25 mM NaN3 to prevent microbial activity, enzyme hydrolysis was initiated by adding 0.25 mL of 2 M buffer (0.1 M final concen- tration) containing enzyme and 2 mM MgC1 2 (see Table 2 for enzyme preparations). Samples were diluted to 5 mL with deionized water and incubated for 16 h overnight at 37°C in a shaking water bath. After incubation, the enzyme reactions were terminated and inorganic P determined by adding 1-mL molybdate reagent as described previously. Phosphodiesterase and phytase caused slight interference with the molybdate reaction; so separate calibration curves were prepared from orthophosphate standards containing the enzymes.
Table 2. The enzyme preparations used to determine phosphatase hydrolyzable P.
Enzyme Type Source Specified activity Sigma
No. Buffer Activity of preparation
unit(s) mL ' Alkaline phosphatase
mucosa 2980 units mg ' protein
(10 mg protein mL ••') P 5521 2 M Tris-HCI,
pH 8.0 1 Phospholipase Ct
(EC 3.1.4.3) Type XI Bacillus cereus 1076 units mg ' protein
(0.9 mg protein mL P 7147 2 M Tris-HCI,
pH 8.0 1 Phosphodiesteraset
(EC 3.1.4.1) Phosphodiesterase 1,
Type IV Crotalus atrox
venom 0.031 units mg ' solid P 4506 2 M Tris-HCI,
pH 8.0 0.016 Phytase
(EC 3.1.3.8) myo-inositol
hexakisphosphate Aspagillus ficuum 3.5 units mg ' solid P 9792 2 M Glycine-HCI,
pH 2.5 0.5
3-phosphohydrolase
t The preparation used in the phosphatase hydrolyzable P assays included alkaline phosphomonoesterase.
TURNER ET AL: ORGANIC P COMPOSITION OF SEMI-ARID U.S. SOILS 1171
Functional classes of organic P compounds were calculated as follows: (i) labile monoester P (hydrolyzed by alkaline phos- phatase); phospholipids (the difference between the P released by phospholipase + alkaline phosphatase and alka- line phosphatase alone); (iii) nucleic acids (the difference between the P released by phosphodiesterase + alkaline phos- phatase and alkaline phosphatase alone); (iv) Inositol hexakis- phosphate (the difference between the P released by phytase and all other treatments). These were grouped into total monoester P (labile monoester P and inositol hexakisphos- phate) and orthophosphate diesters (phospholipids and nu- cleic acids).
Statistical Analysis
Concentrations are expressed on the basis of oven-dried soil (105°C). A correlation matrix (r values) was calculated to investigate relationships between soil properties and P com- pounds, which were then investigated visually by plotting on an x-y scatter graph. Regression models were calculated using least squares linear regression. All analysis was performed using standard procedures in Microsoft Excel (Microtsoft Inc., Redmond, WA).
RESULTS Soil Properties
Soil physical and chemical properties are presented in Table 3. The soils are ranked in ascending order of organic C concentrations, which ranged between 1.98 and 30.69 g C kg - ' soil. Soil pH ranged between 5.2 and 8.2, with most soils being greater than pH 6.5. Clay contents ranged between 2 and 48%, and most soils con- tained significant concentrations of CaCO 3 (<1-480 g kg- 1 soil). Concentrations of oxalate-extractable metals (g kg-' soil) were: Al = 0.26 to 1.21, Fe = 0.20 to 2.36, Mn = 0.11 to 1.07.
Total and Extractable Phosphorus Concentrations
Total P concentrations ranged between 220 and 1210 mg P kg- 1 soil, of which inorganic P constituted between 27 and 76% (Table 4). Organic P concentra-
tions determined by the ignition method were between 18 and 280 mg P kg' soil (3-36% total P). Using organic P determined by the ignition method, the organic C/organic P ratios ranged between 37 and 131 (mean 75).
Between 12 and 45% of the total soil P was recovered by extraction with NaOH-EDTA (Table 4). Of this, between 21 and 178 mg P kg- 1 soil was organic P, repre- senting recoveries of between 23 and 118% (mean 63%) of the total organic P determined by the ignition method. A smaller proportion of the inorganic P was recovered (12-95 % total inorganic P, mean 31%). Using organic P determined by NaOH-EDTA extraction and molybdate colorimetry, the organic C/organic P ratios ranged between 50 and 198 (mean 125).
Extraction with bicarbonate recovered between 1.6 and 13.4% of the total soil P (Table 4). Of this, between 6.5 and 106 mg P kg -1 soil was inorganic P and between 1.7 and 22.8 mg P kg-' soil was organic P. The organic P concentrations represented recoveries of between 1.5 and 12.0% of the total organic P determined by ignition.
Organic Phosphorus Composition of NaOH-EDTA Extracts Determined by
Solution 31P NMR Spectroscopy The NaOH-EDTA extracts contained inorganic or-
thophosphate, orthophosphate monoesters, orthophos- phate diesters, and pyrophosphate (Table 5, Fig. 2). Phosphonates and polyphosphates were not detected in any samples. Inorganic orthophosphate dominated the extracts of all soils, although concentrations determined by solution 31 P NMR spectroscopy were consistently greater than those determined by molybdate colorime- try (Fig. 3). Orthophosphate monoesters were the major group of organic P compounds, constituting between 11 and 130 mg P kg-' soil (10-44% extracted P) (Table 5). Orthophosphate diesters were not detected by solution 31 1) NMR spectroscopy in 11 of the soils, but concentra- tions in the remaining soils ranged between 1.4 and 7.0 mg P kg -1 soil (0.7-2.6% extracted P). Total organic P concentrations detected by NaOH-EDTA extraction and solution 31 P NMR spectroscopy were, therefore,
Table 3. Physical and chemical properties of the 18 western U.S. soils, ranked in order of their organic C concentrations.
Soil type Textural class
soil
2 8 90 1.98 kgg
7 0.26 0.36 0.111. Taunton Sand 8.2 2. Olton Clay loam 17 20 63 7.8 3.37 1 0.46 0.22 0.14
3. Declo Loam 15 27 58 7.9 431 99 0.44 0.32 0.13 4. Warden Very fine sandy loam 10 41 49 8.1 5.35 22 0.80 1.30 0.27 5. Amarillo Fine sandy loam 14 16 70 7.7 5.35 1 0.47 0.31 0.11
6. Portneuf (cony subsoil) Silt loam 15 68 17 8.1 5.49 234 0.40 0.20 0.14
7. Greenleaf Silt loam 23 49 28 7.7 5.88 29 1.09 1.70 0.34
8. Williams Loam 14 21 65 7.1 8.02 4 0.43 0.60 0.29 9. Portneuf (manured subsoil) Silt loam 20 64 16 8.1 8.12 234 0.46 0.27 0.14
10. Portneuf (cony ) Silt loam 26 56 17 7.9 8.23 47 0.75 0.65 0.47
11. Roza Clay loam 22 50 28 6.9 8.81 2 0.70 0.82 0.44 12. Portneuf (manured) Silt loam 23 57 19 8.0 9.11 106 0.62 0.52 0.34
13. Mil!vine Silt loam 15 47 38 8.1 10.39 480 0.74 0.63 0.12
14. Portneuf (native) Silt loam 15 62 23 6.7 13.42 3 0.58 0.92 0.53
15. Brinegar 16. Palouse
19 24
32 58
49 18
5.6 5.2
13.46 18.23
2 <1
0.57 1.21
1.56 2.36
0.20 0.40
17. Labenzo Silt loam 19 41 40 7.6 25.34 48 0.74 0.86 0.23 18. Wahpeton Silty clay 48 37 15 6.4 30.69 6 0.75 2.06 1.07
1172 SOIL SCI. SOC. AM. J., VOL. 67, JULY—AUGUST 2003
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between 11 and 132 mg P kg-' soil (10-47% extracted P). Pyrophosphate was detected in all but four soils (although there were traces in two of those) at concen- trations between 1.1 and 13.5 mg P kg -1 soil (0.5-4.3% extracted P). A trace of a compound with a chemical shift upfield from inorganic orthophosphate at approxi- mately 6.5 ppm was identified in one soil (Greenleaf), which may represent an aromatic orthophosphate dies- ter (Turner et al., 2003).
Phosphatase Hydrolysis of Bicarbonate- Extractable Organic Phosphorus
Concentrations of bicarbonate-extractable organic P hydrolyzed by phosphatase enzymes ranged between 1.4 and 8.4 mg P kg -1 soil, equivalent to between 37 and 87% of the bicarbonate-extractable organic P (Table 6). Of this, between 0 and 4.7 mg P kg-' soil was labile monoester P hydrolyzed by alkaline phosphatase (0-32% bicarbonate organic P) and between 1.2 and 5.8 mg P kg- 1 soil was total monoester P (26-77% bicar- bonate organic P). Between 0 and 2.6 mg P kg' soil was orthophosphate diesters (0-24% bicarbonate or- ganic P). These were dominated by nucleic acids (hy- drolyzed by phosphodiesterase), with only small concen- trations of phospholipids. It should be noted that many of the values reported here were close to or below the limit of detection for the bicarbonate method (approxi- mately 0.2 mg P kg-' soil).
Relationships Between Climate, Soil Properties, and Phosphorus Fractions
Climate and Soil Properties
Organic C concentrations were positively correlated with concentrations of clay and oxalate Fe and Mn, and negatively correlated with soil pH (Table 7). Clay content was positively correlated with oxalate Fe and Mn, and soil pH was negatively correlated with oxalate Fe. However, clay and pH were not correlated. No soil properties were significantly correlated with CaCO 3 concentrations. Organic C was negatively correlated with mean annual temperature and positively correlated with mean annual precipitation when the two Texan soils (Olton and Amarillo) were omitted. Mean annual precipitation and temperature were not correlated, but a weak negative correlation existed between mean annual precipitation and soil pH.
Organic Phosphorus Fractions and Soil Properties
Organic P determined by NaOH-EDTA extraction and solution 31P NMR spectroscopy was positively corre- lated with organic C, clay content, and oxalate metals (Al, Fe, Mn), and negatively correlated with soil pH (Table 8, Fig. 4). Similar relationships were evident for orthophosphate monoesters (which constituted most of the NaOH-EDTA organic P) and orthophosphate dies- ters, although the correlation with clay was stronger with orthophosphate diesters. Pyrophosphate was positively correlated with organic C, clay, and oxalate Mn, but was not significantly correlated with soil pH or oxalate
Soil 1. Taunton Inorganic
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TURNER ET AL: ORGANIC P COMPOSITION OF SEMI-ARID U.S. SOILS 1173
Table 5. Concentrations of functional inorganic and organic P groups in NaOH-EDTA extracts of the 18 western U.S. soils determined by solution 31P NMR spectroscopy. Values in parentheses are the proportion (%) of the total NaOH-EDTA extractable P.
Soil type Inorganic
orthophosphate Pyrophosphate Orthophosphate
P kg Img soil
1. Taunton 92 (90) n.d.t 11 (10) n.d. 11 (10) 2. Olton 51 (70) 3.1 (4.3) 19 (26) n.d. 19 (26) 3. Declo 147 (66) 1.9 (0.9) 69 (31) 3.7 (1.7) 73 (33) 4. Warden 133 (76) 1.4 (0.8) 41 (23) n.d. 41 (23) 5. Amarillo 83 (74) trace 29 (26) n.d. 29 (26) 6. Portneuf (cony subsoil) 89 (77) 2.1 (1.8) 25 (22) n.d. 25 (22) 7. Greenleaf 167 (81) n.d. 35 (17) 3.1 (1.5) 38 (19) 8. Williams 72 (56) 2.7 (2.1) 52 (41) 1.4 (1.1) 54 (42) 9. Portneuf (manured subsoil) 212 (83) 4.7 (1.8) 37 (15) n.d. 37 (15) 10. Portneuf (cony) 167 (77) 3.0 (1.4) 48 (22) n.d. 48 (22) 11. Roza 113 (74) 1.4 (0.9) 39 (25) n.d. 39 (25) 12. Portneuf (manured) 220 (77) 1.9 (0.7) 65 (23) n.d. 65 (23) 13. Millville 164 (73) trace 60 (27) n.d. 60 (27) 14. Portneuf (native) 147 (77) 2.0 (1.1) 42 (22) n.d. 42 (22) 15. Brinegar 147 (69) 1.1 (0.5) 64 (30) 2.1 (1.0) 66 (31) 16. Palouse 165 (58) 8.3 (2.9) 107 (38) 3.3 (1.2) 111 (39) 17. Labenzo 175 (54) 13.5 (4.2) 130 (40) 2.2 (0.7) 132 (41) 18. Wahpeton 133 (49) 11.7 (4.3) 120 (44) 7.0 (2.6) 127 (47)
t n.d., not detected.
Fe. No relationships were evident between CaCO 3 con- centrations and NaOH-EDTA extractable organic P fractions. Soil organic P determined by ignition was positively correlated with organic C and clay, and nega- tively correlated with mean annual temperature, but the correlations were weaker than those for organic P
11111111111 1111 111111 111111
20 10 0 -10
-20
Chemical shift Fig. 2. Solution uP NMR spectra of NaOH-EDTA extracts of the
Taunton soil (smallest organic C concentration) and Wahpeton soil (largest organic C concentration), indicating the compounds present in the extracts.
determined by NaOH-EDTA extraction and solution 3 'P NMR spectroscopy (Table 7).
Bicarbonate-extractable organic P was positively cor- related with organic C, oxalate Al and Fe, and organic P extracted by NaOH-EDTA, and was negatively corre- lated with soil pH (Table 8, Fig. 4). All phosphatase hydrolyzable P fractions were negatively correlated with soil pH, and orthophosphate diesters were correlated with oxalate Al and Fe (Table 8). Concentrations of bicarbonate-extractable monoester and diester P were also correlated with the respective NaOH-EDTA ex-
0 50 100 150 200 250
Inorganic orthophosphate by 31 P NMR (mg P kg- 1 soil)
Fig. 3. Relationship between inorganic orthophosphate (mg P soil) in NaOH-EDTA extracts of the 18 western U.S. soils determined by molybdate colorimetry and solution ' 1P NMR spec- troscopy. The regression model is described by the equation: (Ortho- phosphate by molybdate colorimetry] = 0.899 ± 0.032[orthophos- phate by 31P NMR] -6.023 ± 4.572; IV = 0.98, F = 815, P <
0.0001, n = 18)
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tractable organic P classes (r = 0.65 and 0.68, respec- tively, P < 0.05).
Organic Phosphorus Fractions and Climate
Organic P fractions determined by NaOH-EDTA ex- traction and solution "P NMR spectroscopy were signif- icantly correlated with mean annual temperature and precipitation, although in some cases the relationships were significant only when outlying soils were omitted (Table 8, Fig. 4). Mean annual temperature was nega- tively correlated with the concentrations and propor- tions of orthophosphate monoesters and diesters ex- tracted with NaOH–EDTA, and with pyrophosphate when the two Texan soils were omitted (Table 8, Fig. 4). In contrast, mean annual precipitation was positively correlated with all NaOH–EDTA extractable organic P fractions, although again the relationships were only statistically significant for total NaOH–EDTA organic P and orthophosphate monoesters when the Texan soils were omitted.
All bicarbonate-extractable organic P fractions were negatively correlated with mean annual temperature, but only the correlation with orthophosphate diesters was statistically significant. However, the correlations with bicarbonate-extractable organic P and labile monoes- ters were significant when a single outlying soil (Pa- louse) was omitted (Table 8, Fig. 4). The proportions of labile monoesters and orthophosphate diesters in the bicarbonate extracts were also negatively correlated with mean annual temperature, but were not statistically significant. Mean annual precipitation was positively correlated with all bicarbonate-extractable organic P fractions, but was only statistically significant for bicar- bonate-extractable organic P. However, the relation- ships were statistically significant for orthophosphate monoesters and diesters when the Texan soils were omitted.
Regression Analysis
Soil organic P extracted by NaOH–EDTA and deter- mined by solution "P NMR spectroscopy was predicted by a model including mean annual temperature and precipitation, and a similar model described the concen- tration of orthophosphate monoesters (Table 9). Both models were improved slightly, but not statistically sig- nificantly, by including clay content (R2 = 0.670 and 0.655, respectively). Bicarbonate-extractable organic P was predicted by a model containing soil pH and oxalate Al (Table 9). This model was improved slightly by in- cluding mean annual precipitation and temperature ei- ther alone (R2 = 0.84) or together (R2 = 0.86), but in both cases the additional variables were not statistically significant (P > 0.10).
DISCUSSION Cultivated soils usually contain lower organic P con-
centrations than equivalent uncropped soils, because tillage increases aeration, exposing organic matter to a more vigorous microbial attack (Anderson, 1980). How-
TURNER ET AL: ORGANIC P COMPOSITION OF SEMI-ARID U.S. SOILS
1175
Table 7. Correlation coefficients for relationships between physical and chemical properties of thel8 western U.S. soils.
Organic C PH Clay CaCO3 Oxalate Al Oxalate Fe Oxalate Mn Mean annual temperature
Mean annual precipitation
pH -0.57** Clay 0.72*** NSt CaCO, NS NS NS Oxalate Al NS NS NS NS Oxalate Fe 0.625* -0.75*** 0.56* NS 0.80*** Oxalate Mn 0.69*** NS 0.83*** NS NS 0.62** Mean annual temperature -0.60** NS NS NS NS NS NS Mean annual precipitation -0.52* NS NS NS NS NS NS Total P NS NS NS NS 0.47* NS NS NS -0.54* Total inorganic P NS NS NS NS NS NS NS NS -0.69*** Total organic P 0.80*** NS 0.57* NS NS NS NS -0.60** NS Residual P NS -0.62** NS NS 0.70*** 0.79*** NS NS NS
* Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *5* Significant at the 0.001 probability level. tNS, not significant.
r = 0.73** when two outlying soils from Texas were omitted.
ever, the western U.S. soils studied here contained rela- tively low concentrations of organic P compared with cultivated soils from other climates and containing a wider range of soil properties (Anderson, 1980; Con- dron et al., 1990a; Guggenberger et al., 1996). For exam- ple, Condron et al. (1990a) reported organic P concen- trations of 122 to 212 mg P kg -1 soil in Canadian soils that had been under arable cropping for at least 70 yr.
Several interacting climate factors and soil properties are likely to contribute to the low organic matter con- centrations in the soils studied here. Climate seemed to exert an important influence, as suggested elsewhere. For example, Sumann et al. (1998) reported that the proportions of orthophosphate monoesters and diesters in NaOH-NaF extracts of North American Great Plains soils correlated strongly with mean annual temperature and precipitation, while McKercher and Anderson (1968) suggested that differences in the concentrations of inositol phosphates between British and Canadian soils were linked to differences in climate. We found strong negative correlations between soil organic P con- centrations and mean annual temperature. Decreasing organic P concentrations with increasing temperature was also reported for organic P in soils of South Dakota (Westin and Buntley, 1967), which probably relate to accelerated rates of microbial and enzyme activity at higher temperatures (Eid et al., 1951; Spier and Ross, 1978). The positive relationship between soil organic P and mean annual precipitation is more complex, because increases in precipitation can increase or decrease soil organic P concentrations. In wet soils, elevated soil moisture can suppress microbial activity by reducing aeration, which can lead to the accumulation of phos- phonates in cool moist soils (Tate and Newman, 1982). However, in drier regions, such as much of the western USA, increasing soil moisture increases biological pro- ductivity, which in turn increases organic matter inputs to the soil (Anderson, 1980). Wetter soils also experi- ence shorter or less intense dry periods, reducing the impact of soil drying on organic P solubility (Turner and Haygarth, 2001). The two Texan soils were consistent outliers in the relationships between soil organic P frac- tions and precipitation, which may reflect the combina-
tion of relatively high temperature and precipitation in that region (Table 1).
The strong correlations between organic P and C con- centrations and the soil pH, clay and amorphous Fe, - suggest the importance of adsorption and stabilization of organic matter in these soils. Similar correlations were reported for 168 benchmark soils from the USA with a wide range of physical and chemical properties (organic C 2-250 g C kg-' soil, pH 3.3-8.5, CaCO 3 5-550 g kg- 1 soil) (Tiessen et al., 1984). Clays stabilize organic matter by providing reactive surfaces, which in turn provide physical protection from microbial attack. The stability of most phosphate esters increases below pH 5 (Anderson and Arlidge, 1962; Greaves and Wilson, 1969), and microbial activity is also suppressed in acidic conditions (Anderson, 1980). Thus, the soil environ- ment exerts a strong control on organic matter accumu- lation. In contrast, the often substantial concentrations of CaCO3 did not appear to exert a control on the accumulation of organic C or organic P compounds in these soils. Tiessen et al. (1984) reported a similar result, although Sen Gupta and Cornfield (1962) reported that organic P accumulated in calcareous soils with increas- ing concentrations of CaCO3 .
Most of the organic P in the western U.S. soils studied here was orthophosphate monoesters, with smaller con- centrations of orthophosphate diesters, which is consis- tent with the behavior of these functional classes of organic P in the soil. Orthophosphate monoesters are likely to be mainly inositol phosphates. These com- pounds constitute only small inputs to the soil in plant and microbial residues, but react strongly because of their high charge density (Turner et al., 2002b). In con- trast, inputs of orthophosphate diesters to the soil are quantitatively greater than those of inositol phosphates, but their weak adsorption, especially in soils of pH greater than 5, makes them susceptible to microbial degradation (Greaves and Wilson, 1969). This differen- tial behavior means that orthophosphate diesters are preferentially degraded, for example during the decline in soil organic P associated with long-term cultivation (Condron et al., 1990a). Thus, orthophosphate monoes- ters are preferentially stabilized and accumulate to form
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1176 SOIL SCI. SOC. AM. J., VOL 67, JULY-AUGUST 2003
the dominant class of organic P in most soils, including the western U.S. soils of the current study.
Pyrophosphate was also present in most soils and appeared to be controlled by similar mechanisms to both orthophosphate monoesters and diesters. Inor- ganic polyphosphates appear to originate from micro- bial activity and are rapidly degraded in soils, but pyro- phosphate (an inorganic polyphosphate with chain length n = 2) can become stabilized in soils and remain for many months (Blanchar and Hossner, 1969). Therefore, it is unclear whether the presence of pyrophosphate reflects microbial activity or the potential for stabiliza- tion in the soil.
Concentrations of bicarbonate-extractable organic P were controlled primarily by soil pH rather than climate or clay content. A similar strong relationship was re- ported for a range of UK pasture soils, in which bicar- bonate-extractable organic P concentrations increased markedly in soils less than pH 5 (Turner and Haygarth, 2003). Bicarbonate-extractable organic P has been sug- gested to be an active pool of soil organic P, and proba- bly represents compounds that are readily exchangeable from the soil (Bowman and Cole, 1978). However, there is little information on the chemical composition of bi- carbonate-extractable organic P with which to infer its biological availability. In the current study, bicarbonate- extractable organic P was of a similar composition to the total soil organic P determined by NaOH—EDTA extraction and 31 P NMR spectroscopy, which agrees with the organic P composition of Canadian arable soils de- termined using solution 31 P NMR analysis of bicarbon- ate and NaOH extracts (Zhang et al., 1999). The main difference between the composition of bicarbonate and NaOH—EDTA extractable organic P in the western U.S. soils was the greater proportions of diesters in bicarbon- ate extracts. This probably reflects the greater sensitivity of the phosphatase hydrolysis technique, which can de- tect lower concentrations of orthophosphate diesters than solution 31P NMR spectroscopy, plus the rapid deg- radation of some orthophosphate diesters (RNA and some phospholipids) in alkaline solution, which will lead to the underestimation of orthophosphate diesters in most soils (Turner et al., 2003).
Bicarbonate-extractable organic P concentrations in these western U.S. soils were low, but large proportions were hydrolyzable by common soil phosphatase en- zymes and were, therefore, potentially bioavailable. Similar results were reported for volcanic soils of the Canary Islands, in which between 33 and 93% of the bicarbonate-extractable organic P was hydrolyzed by a nonspecific acid phosphatase from wheat (Negrin et al., 1995). These large proportions of phosphatase-hydro- lyzable organic P may partly explain discrepancies in correlations between plant-available P pools and crop response, and support the suggestion that soil organic P could account for the difficulties encountered in de- termining soil P fertility using inorganic P determina- tions alone (Tiessen et al., 1984). Our results should be considered with caution, however, because analytical error was large relative to the small concentrations of phosphatase hydrolyzable P in these soils. Further, the
0 0
0
O
Organic C (g C kg-1 soil)
6 7 8
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00PD 0
TURNER ET AL: ORGANIC P COMPOSITION OF SEMI-ARID U.S. SOILS
1177
O
O
O 0 0 O
°0 0 otz CSO
0 8 0O 00 0
Fig. 4. Relationships between soil properties, climate variables (mean annual precipitation [min) and temperature I°C1) and organic P determined by bicarbonate extraction and molybdate colorimetry, and by NaOH-EDTA extraction and solution uP NMR spectroscopy (mg P kg - ' soil). Outlying soils not included in the calculation of correlation coefficients are identified as shaded circles.
results are in direct contrast to studies of Japanese and Australian arable soils, in which only small proportions of bicarbonate-extractable organic P were hydrolyzed by phosphatase and phytase (Otani and Ae, 1999; Hayes et al., 2000). Bicarbonate-extractable organic P concen- trations can also vary considerably in response to soil drying (Turner and Haygarth, 2003), so the results for air-dried soils may not readily translate to field condi- tions. However, the soils studied here are dry for much of the year, and regularly experience temperatures at the surface in excess of 50°C during the summer, sug- gesting that mild drying is unlikely to greatly influence organic P solubility.
Conventional techniques for measuring total soil or- ganic P suffer from various errors that can severely limit their accuracy (Condron et al., 1990b). The ignition method overestimates organic P in most soils because high temperature ignition alters the solubility of mineral P compounds (Williams et al., 1970). This in turn limits confidence in extraction techniques, because there is no means of quantifying the recovery of organic P (Bow- man and Moir, 1993). The current study highlights a further source of error in the measurement of extracted organic P by colorimetry, because inorganic orthophos- phate in alkaline extracts is underestimated by molyb- date reaction. This has been noted elsewhere (Bedrock et al., 1994; Hupfer and Gachter, 1995; Guggenberger
et al., 1996) and probably indicates the presence of or- thophosphate-metal-humic complexes not detected by molybdate reaction. In the current study, organic P con- centrations determined by NaOH-EDTA extraction and solution 31P NMR spectroscopy appeared closer to the likely true values than those determined by ignition, because extracted organic P was more strongly corre- lated with organic C, and gave slightly greater organic C/organic P ratios (Condron et al., 1990b; Bowman and Moir, 1993). The degradation of RNA and phospho- lipids in NaOH-EDTA does not affect the determina- tion of total organic P determined by NaOH-EDTA extraction and solution 31P NMR spectroscopy, because these compounds degrade to orthophosphate monoes- ters rather than inorganic orthophosphate (Turner et al., 2003). Taken together, these results suggest that NaOH-EDTA extraction and solution 31 P NMR spec- troscopy provides the most accurate estimate of soil organic P.
CONCLUSIONS The organic P extractable by NaOH-EDTA in irri-
gated agricultural soils of the western USA with a wide range of textural and chemical properties was mainly orthophosphate monoesters, with smaller concentra- tions of orthophosphate diesters and pyrophosphate.
Table 9. Regression models describing the influence of climate and soil properties on (1) soil organic P determined by NaOH-EDTA extraction and solution 31P NMR spectroscopy, (2) orthophosphate monoesters by the same technique, and (3) bicarbonate-extractable organic P.
Y variate variate Gradient Xi X, variate Gradient 12 Intercept R2 F value P value
1. NaOH-EDTA organic P, mg P kg ' soil
2. NaOH-EDTA orthophosphate •monoesters, mg P kg ' soil
3. Bicarbonate organic P, mg P kg ' soil
Mean annual temperature, °C
Mean annual temperature, °C
0.15 ± 0.05
0.15 ± 0.05
5.99 ± 2.69
91.8 25.7
88.9 ± 25.0
36.3 ± 6.25
SOIL SCI. SOC. AM. J., VOL. 67, JULY -AUGUST 2003
Phosphatase hydrolysis of bicarbonate-extractable or- ganic P revealed a similar composition, and suggested the potential biological availability of this readily ex- changeable organic P pool. Strong correlations between soil organic P, climate, and soil properties suggested that equilibrium levels of organic P in western U.S. soils reflect a balance between the physical protection offered by the soil matrix, and the suitability of the environment for biological productivity. In contrast, the presence of often large concentrations of CaCO 3 ap- peared to have little influence on soil organic P concen- trations.
ACKNOWLEDGMENTS
NMR analysis was performed at the Stanford Magnetic Resonance Laboratory with support funding from the Stan- ford University School of Medicine. We thank Dr Corey Liu, Susie Hansen, and Paula Jolley for their contribution, and the many others who helped to collect the soil samples used in this study.
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